The present invention relates to a fluorescence analyzer which operates rapidly and provides automatic measurements of a large number of samples.
In the research environment fluorescence measuring apparatus can be used for organic and inorganic identifications of compounds and for the study of molecular structures. It is applied to the study of enzyme mechanisms and kinetics, polypeptide derivative and synthesis studies, polymer properties, development of new dye and stain techniques, biological pathways and interactions.
In clincial analysis such apparatus is used for measurements in immunoserology, microbiology, toxicology, clinical chemistry, histopathology, and coagulation.
In the industrial and agricultural area fluorescence measuring apparatus can be used for toxic assays and contamination studies. This apparatus can be used for quality control and contamination studies in food processing and provides measurements appropriate to chemical applications useful to toxic studies, mutagenic and carcinogenic assays, polymer chemistry, purity analysis as well as chromotography techniques and organic and inorganic molecular analysis.
In the fermentation area it is used in enzyme immobilization and purity analysis. In the pharmaceutical area its applications are enzyme systems analysis, drug effect studies, amino acid analysis and pharmacological monitoring. In veterinary medicine it can be used for diagnostic monitoring, toxin studies, monitoring for pharmacology studies and breeding verifications.
In environmental applications, it can be used for inorganic and organic pollution monitoring, toxin studies, microbial assays and carcinogenic assays, and in the petrochemical industry its applications include carcinogenic and contamination studies, refining assays and quality control.
One powerful discipline is the immunalogical studies. In recent years, new immunological methods have been developed in which plates having a plurality of wells for holding samples (one type of such carrier is the "MICROTITER" plate); and are coated with the specific antigen, incubated further with the specific antibody with or without the addition of an "unknown" amount of the same antigen, and subsequently, a second antibody, directed against the first antibody, is added. The latter antibody is conjugated to an enzyme such as peroxidase or alkaline phosphatase. The amount of enzyme immobilized on the microtiter plate is determined by the addition of the proper substrate. The colorimetric reaction is measured by a spectrophotometer which is designed to accomodate microtiter plates.
An alternative method is to coat the plates first with the specific antibody, then add enzyme-labeled antigen as a second layer plus an "unknown" antigen, and finally, add the enzyme substrate as described above. The same type of measurement is then made by the spectrophotometer. This measurement method is called the enzyme immunoassay (EIA) or the enzyme-linked immunosorbent assay (ELISA).
The EIA and ELISA detection methods are highly sensitive and compare with other chemical methods such as high-pressure liquid chromatography and radioimmunoassay methods used in the prior art. The EIA assay is as specific as the antibody used. If the antibody is extremely specific, then the assay will also be extremely specific. For example, a highly specific antibody will not cross-react with other antigens, e.g., antibodies specific for native DNA will not cross react with single-stranded DNA.
The EIA assay has been used to detect hormones and in many instances has replaced with equal or better sensitivity the radioimmunoassay methods for hormone detection. The EIA assay also is used to detect immunoglobulins, carcinogenic antigens, infectious disease and viral infections. Rubella antigens, EB virus, measles, herpes viruses and others are examples of viral antigens already detected by EIA. EIA assay methods have found utilization in veterinary and agricultural applications. Future applications will involve the detection of auto-immune diseases, transplantation antigens, and virtually any compound which is being assayed currently by chemical methods or by radioimmunoassay.
Even though the EIA assay method has provided highly sensitive and rapid measurement capabilities, the current instruments used are essentially colorimeters, and thus limit the choice of substrates to those that produce color reactions. Even though other substrates are available which may be many times more sensitive, no instruments were available which could use such substrates. By using a fluorescent substrate a further increased sensitivity of manyfold over that of using a colorimetric substrate, has been observed. In some cases, measurement of antigen levels 1,000 fold smaller than can now be detected on the same microtiter plate, could be detected using a fluorescence substrate. The present invention is an apparatus which can measure the fluorescence of the enzyme substrate complex directly in the microtiter plates.